Cell concentration methods and devices for use in automated bioreactors

ABSTRACT

The present disclosure provides cassettes for use in automated cell engineering systems that include cell concentration filters for reducing fluid volume of a cell sample during or following automated processing. The disclosure also provides methods of concentrating a cell population, as well as automated cell engineering systems that can utilize the cassettes and carry out the methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims benefit of U.S. Provisional PatentApplication No. 62/803,219, filed Feb. 8, 2019, the disclosure of whichis incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present disclosure provides cassettes for use in automated cellengineering systems that include cell concentration filters for reducingfluid volume of a cell sample during or following automated processing.The disclosure also provides methods of concentrating a cell population,as well as automated cell engineering systems that can utilize thecassettes and carry out the methods.

BACKGROUND OF THE INVENTION

As anticipation builds about accelerated clinical adoption of advancedcell therapies, more attention is turning to the underlyingmanufacturing strategies that will allow these therapies to benefitpatients worldwide. While cell therapies hold great promise clinically,high manufacturing costs relative to reimbursement present a formidableroadblock to commercialization. Thus, the need for cost effectiveness,process efficiency and product consistency is driving efforts forautomation in numerous cell therapy fields.

Automation of various processes is involved in producing cellpopulations for therapy. This includes integration of cell activation,transduction and expansion into a commercial manufacturing platform, forthe translation of these important therapies to the broad patientpopulation.

It is often necessary to reduce the volume of a cell population, eitherduring automated processing, or prior to a final output from theautomated system. What is needed is a process by which a cellular samplecan be concentrated, i.e., the volume of the sample reduced, eitherduring the automation or prior to a sample output.

SUMMARY OF THE INVENTION

In some embodiments, provided here is a cassette for use in an automatedcell engineering system, comprising a cell culture chamber, a pumpingsystem fluidly connected to the cell culture chamber, a tangential flowfilter fluidly connected to the pumping system, wherein the pumpingsystem provides a retentate flow to the tangential flow filter andwherein a permeate flow of the tangential flow filter is controlled by aflow controller, and a cellular sample output fluidly connected to thetangential flow filter.

In further embodiments, provided herein is a cassette for use in anautomated cell engineering system, comprising a cell culture chamber, apumping system fluidly connected to the cell culture chamber, atangential flow filter fluidly connected to the pumping system, whereinthe pumping system provides a retentate flow to the tangential flowfilter and wherein a permeate flow of the tangential flow filter iscontrolled by a flow controller, a satellite volume connected to thetangential flow filter, a fluidics pathway for recirculating theretentate flow back through the tangential flow filter, a fixed volumewaste collection chamber fluidly connected to the tangential flowfilter, and a cellular sample output fluidly connected to the tangentialflow filter.

In additional embodiments, provided herein is a method of reducing avolume of a cellular sample during automated processing, the methodcomprising introducing a cellular sample into a tangential flow filterhaving a retentate flow and a permeate flow, wherein the permeate flowis controlled by a flow controller, passing the cellular sample throughthe retentate flow of the tangential flow filter, removing volume fromthe cellular sample via the permeate flow to a fixed volume wastecollection chamber, and collecting the cellular sample having thereduced volume.

In still further embodiments, provided herein is an automated cellengineering system, comprising an enclosable housing, a cassettecontained within the enclosable housing, the cassette comprising a cellculture chamber, a pumping system fluidly connected to the cell culturechamber, a tangential flow filter fluidly connected to the pumpingsystem, wherein the pumping system provides a retentate flow to thetangential flow filter and wherein a permeate flow of the tangentialflow filter is controlled by a flow controller, and a cellular sampleoutput fluidly connected to the tangential flow filter, and a userinterface for receiving input from a user.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows various steps that can be performed with a cassette of anautomated cell engineering system, as described in embodiments hereof.

FIG. 2A shows an exemplary cassette in accordance with embodimentshereof.

FIG. 2B shows an exemplary tangential flow filter for use in thecassettes, systems and methods described herein.

FIG. 2C shows exemplary flow controllers for use with the tangentialflow filters as described herein.

FIGS. 3A and 3B show images of an automated cell engineering system inaccordance with embodiments hereof.

FIG. 4 shows a lab space containing exemplary cell engineering systemsas described in embodiments hereof.

FIG. 5 shows a flowpath for cell concentration in a cassette of anautomated cell engineering system as described in embodiments hereof.

FIG. 6A-6B show the effect of serum on tangential flow filtration, inaccordance with embodiments hereof.

FIGS. 7A-7C show the use of permeate control to reduce the clogging ofthe tangential flow filter in accordance with embodiments hereof.

FIGS. 8A-8B show volume reduction of peripheral blood mononuclear cells(PBMC) using tangential flow filtration in accordance with embodimentshereof.

FIGS. 9A-9D show permeate pump optimization during tangential flowvolume reduction of PMBCs in accordance with embodiments hereof.

FIG. 10A shows cell recovery post tangential flow filtration, inaccordance with embodiments hereof.

FIG. 10B shows cell viability pre- and post-tangential flow filtration,in accordance with embodiments hereof.

FIG. 11 shows CD4+ and CD8+ expression in control and TFF cellsuspensions.

DETAILED DESCRIPTION OF THE INVENTION

It should be appreciated that the particular implementations shown anddescribed herein are examples and are not intended to otherwise limitthe scope of the application in any way.

The published patents, patent applications, websites, company names, andscientific literature referred to herein are hereby incorporated byreference in their entirety to the same extent as if each wasspecifically and individually indicated to be incorporated by reference.Any conflict between any reference cited herein and the specificteachings of this specification shall be resolved in favor of thelatter. Likewise, any conflict between an art-understood definition of aword or phrase and a definition of the word or phrase as specificallytaught in this specification shall be resolved in favor of the latter.

As used in this specification, the singular forms “a,” “an” and “the”specifically also encompass the plural forms of the terms to which theyrefer, unless the content clearly dictates otherwise. The term “about”is used herein to mean approximately, in the region of, roughly, oraround. When the term “about” is used in conjunction with a numericalrange, it modifies that range by extending the boundaries above andbelow the numerical values set forth. In general, the term “about” isused herein to modify a numerical value above and below the stated valueby a variance of 20%.

Technical and scientific terms used herein have the meaning commonlyunderstood by one of skill in the art to which the present applicationpertains, unless otherwise defined. Reference is made herein to variousmethodologies and materials known to those of skill in the art.

In embodiments, provided herein are cassettes for use in an automatedcell engineering system. FIG. 1 shows an exemplary cassette 102, inwhich various processes can be carried out in an enclosed, automatedsystem that allows for production of various cellular samples andpopulations. Such processes can include activating, transducing,expanding, concentrating, washing, and collecting/harvesting steps.

As described herein, the cassettes and methods are utilized and carriedout in a fully enclosed automated cell engineering system 300 (see FIGS.3A, 3B), suitably having instructions thereon for performing steps suchas, activating, transducing, expanding, concentrating, and harvesting.Cell engineering systems for automated production of, for examplegenetically modified immune cells, including CAR T cells, are describedin U.S. patent application Ser. No. 16/119,618, filed Aug. 31, 2018 (thedisclosure of which is incorporated by reference herein in itsentirety), and are also called automated cell engineering system,COCOON™, or COCOON™ system herein.

For example, a user can provide an automated cell engineering systempre-filled with a cell culture and reagents (e.g., an activationreagent, a vector, cell culture media, nutrients, selection reagent, andthe like) and parameters for the cell production (e.g., starting numberof cells, type of media, type of activation reagent, type of vector,number of cells or doses to be produced, and the like). The automatedcell engineering system is able to carry out the various automatedmethods, including methods of producing genetically modified immune cellcultures, including CAR T cells, without further input from the user. Insome embodiments, the fully enclosed automated cell engineering systemminimizes contamination of the cell cultures by reducing exposure of thecell culture to non-sterile environments. In additional embodiments, thefully enclosed automated cell engineering system minimizes contaminationof the cell cultures by reducing user handling of the cells.

As described herein, the automated cell engineering systems 300 suitablyinclude a cassette 102. Thus, in embodiments, provided herein is acassette for use in an automated cell engineering system. As used hereina “cassette” refers to a largely self-contained, removable andreplaceable element of a automated cell engineering system that includesone or more chambers for carrying out the various elements of themethods described herein, and suitably also includes one or more of acell media, an activation reagent, a wash media, etc.

FIG. 2A shows an exemplary cassette 102 for use in an automated cellengineering system. In embodiments, cassette 102 includes a cellularsample input 202. Cellular sample input 202 is shown in FIG. 2A as avial or chamber in which a cellular sample can be placed prior tointroduction or loading into cassette 102. In other embodiments,cellular sample input 202 can simply be a sterile-locking tubing (forexample a luer lock tubing connection or the like) to which a syringe ora cell-containing bag, such as a blood bag, can be connected.

Cassette 102 further includes a cell culture chamber 206. Examples ofthe characteristics and uses of cell culture chamber 206 are describedherein. Cassette 102 also includes a pumping system 520 (see FIG. 5 forexemplary location in the flowpath) fluidly connected to cell culturechamber 206.

As used herein, “fluidly connected” means that one or more components ofa system, such as components of cassette 102, are connected via asuitable element that allows for fluids (including gasses and liquids)to pass between the components without leaking or losing volume.Exemplary fluid connections include various tubing, channels andconnections known in the art, such as silicone or rubber tubing, luerlock connections, etc. It should be understood that components that arefluidly connected can also include additional elements between each ofthe components, while still maintaining a fluid connection. That is,fluidly connected components can include additional elements, such thata fluid passing between the components can also pass through theseadditional elements, but is not required to do so.

Pumping system 520 is suitably a peristaltic pump system, though otherpumping systems can also be utilized. A peristaltic pump refers to atype of positive displacement pump for pumping a fluid. The fluid issuitably contained within a flexible tube fitted inside a pumpcasing—often circular. A rotor with a number of “rollers”, “shoes”,“wipers”, or “lobes” attached to the external circumference of the rotorcompresses the flexible tube. As the rotor turns, the part of the tubeunder compression is pinched closed (or “occludes”) thus forcing thefluid to be pumped to move through the tube. Additionally, as the tubeopens after the passing of the cam (“restitution” or “resilience”) fluidflow is induced to the pump. This process is called peristalsis and isused to move fluid through the flexible tube. Typically, there are twoor more rollers, or wipers, occluding the tube, trapping between them abody of fluid. The body of fluid is then transported toward the pumpoutlet.

Cassette 102 also includes a tangential flow filter 204 fluidlyconnected to the pumping system. FIG. 2B shows an exemplary tangentialflow filter. FIG. 2C shows a schematic of an interior of a tangentialflow filter. Tangential flow filtration, also known as crossflowfiltration, is a filtration system or process where a feed, inlet orinput fluid stream (250 in FIG. 2C) passes parallel to a membrane faceas one portion passes through, and out of the membrane (permeateflow—252 in FIG. 2C) while the remainder (retentate flow—254 in FIG. 2C)passes within the membrane and can be recirculated back to the input,becomes concentrated, can ultimately be passed to a storage or output.

Tangential flow filter 204 is suitably comprised of a series of hollowfiber membranes (though a single fiber can also be used), into which asolution is fed. The retentate flow passes within the hollow fiber,retaining cells within the solution inside of the fiber membrane, whileexcess volume passes through the fiber membrane and out into thepermeate flow. This reduces the volume of the total cellular sample,resulting in a concentration of the cellular sample. The membranes aresuitably provided in the form of a self-contained apparatus, which caninclude a flow controller 258.

As described herein, with reference to FIG. 2C, pumping system 520provides retentate flow 254 to tangential flow filter 204, whilepermeate flow 252 of the tangential flow filter is controlled by a flowcontroller 258. “Flow controller” as used herein refers to a valve,constriction device, flow diverter, pump mechanism, fluidics—includingvarious tubing set-ups, or other mechanisms, to control the amount offluid that leaves the fiber membrane of the tangential flow filter andenters the permeate flow. Flow controller 258 in FIG. 2C is providedsimply to illustrate the inclusion of a mechanism for controlling theamount of permeate flow 252, and is does not indicate the structure ofthis mechanism.

In exemplary embodiments, flow controller 258 is a flow restrictor 260.“Flow restrictor” refers to a valve, gradually narrowing tubing, orconstriction device, to control the amount and rate of permeate flow 252exiting the tangential flow filter. Flow restriction 260 is placeddownstream of tangential flow filter 204, so that the control ofpermeate flow occurs after exciting the membranes of tangential flowfilter 204. Flow restrictor 260 is shown in FIG. 2C for illustrativepurposes only, and the location and workings of flow restrictor 260 arenot to be limited by the representation in FIG. 2C. A person of ordinaryskill the art will readily appreciate the various ways that the flowrestrictor can be used to control the amount and rate of permeate flow252. Suitably, flow restrictor 260 is placed adjacent an end 262 oftangential flow filter 204 (see FIG. 2B), to restrict the amount andrate of permeate flow 252.

In further embodiments, flow controller 258 is an additional pumpingsystem that can be set up to control and restrict (or increase) theamount and rate of permeate flow 252.

In still further embodiments, flow controller 258 is a system having aplurality of tubing that can also be orientated and placed withincassette 102 to provide desired control (restriction or increase) of theamount and rate of permeate flow 252.

In embodiments, cassette 102 further includes one or more fluidicspathways suitably connected to the cell culture chamber (see insidecassette 102 in FIG. 2A). Also included in cassette 102 is a cellularsample output 208 fluidly connected to cell culture chamber. Thecassette 102 also suitably includes a cellular sample output 208 fluidlyconnected to tangential flow filter 204.

As described herein, cellular sample output 208 can be utilized toharvest the cells following the various automated procedures for eitherfurther processing, storage, or potential use in a patient. Cellularsample output 208 can also be a sample port 220, as described herein,that allows a cellular sample to be removed from the cassette, forexample for transduction such as electroporation, and then returned tothe cassette for further automated processing. Examples of fluidicspathways include various tubing, channels, capillaries, microfluidicselements, etc., that provide nutrients, solutions, etc., to the elementsof the cassette, as described herein. Cellular sample output 208 canalso simply be the output of the tangential flow filter, which is thenfluidly connected to another section or portion of cassette 102 asdescribed herein.

In embodiments, cassette 102 explicitly excludes a centrifuge before orfollowing tangential flow filter 204. It has been determined thatthrough the use of the various cell separation filters and methodsdescribed herein, additional cellular separation via centrifugationprocedures and the use of a centrifuge is not required. In embodiments,however, an additional filtration system, such as a column filtration,and/or magnetic filtration system, can be utilized.

In exemplary embodiments, tangential flow filter 204 includes a membranewhich has a pore size of about 0.40 μm to about 0.80 μm and a fiberdiameter of about 0.5 mm to about 0.9 mm. In embodiments, the pore sizeof tangential flow filter 204 is about 0.2 μm to about 1.0 μm, or about0.3 μm to about 0.9 μm, about 0.4 μm to about 0.8 μm, about 0.5 μm toabout 0.7 μm, about 0.6 μm to about 0.7 μm, or about 0.40 μm, about 0.45μm, about 0.50 μm, about 0.55 μm, about 0.60 μm, about 0.65 μm, about0.70 μm, about 0.75 μm, or about 0.80 μm. In embodiments, the fiberdiameter is about 0.30 mm to about 1.2 mm, suitably about 0.40 mm toabout 1.0 mm, about 0.50 mm to about 0.90 mm, about 0.60 mm to about0.80 mm, about 0.70 mm to about 0.80 mm, or about 0.60 mm, about 0.65mm, about 0.70 mm, about 0.75 mm, about 0.80 mm, about 0.85 mm, or about0.90 mm.

Suitably, tangential flow filter 204 comprises about 15-20 fibers,suitably 18 filters, having a total length of the lumen of the fibers ofbetween about 10-20 cm, suitably about 10-15 cm, or about 13 cm. Thesurface area of the fibers is on the order of about 40-70 cm², moresuitably about 50-60 cm², or about 57 cm². In embodiments, a relativelyhigh surface area, large pore size membrane is desired for use intangential flow filter 204.

Exemplary materials for use in tangential flow filter 204 includepolymers, including but not limited to, poly(ether sulfone),poly(acrylonitrile) and poly(vinylidene difluoride), cellulose esters,poly(sulfone). Exemplary tangential flow filters include those availablefrom SPECTRUM LABS®, including MICROKROS® and MIDIKROS® filters, andmodifications thereof to fit inside a desired cassette. In embodiments,the material is a modified poly(ether sulfone).

In further embodiments, a coating can be applied to the surface of thetangential flow filter. Suitably, this coating can help to reduce oreliminate fouling on the surface of tangential flow filter 204.Exemplary non-fouling coatings include, for example, phospholipidcoatings, polymeric coatings, such as poly(vinyl alcohol) (PVA),poly(ethylene glycol) coatings, etc. Additional surface coatings canalso be applied to the tangential flow filter to provide stability,increased or decreased flow, or other desired characteristics.

In further embodiments, additional pre- and post-filters (i.e., beforeor after the tangential flow filter) can also be utilized in thecassettes and methods described herein. For example, a magneticseparation process can be utilized to further eliminate and separateundesired cells and debris from a cell population. In such embodiments,a magnetic bead or other structure, to which a biomolecule (e.g.,antibody, antibody fragment, etc.) has been bound, can interact with atarget cell. Various magnetic separation methods, including the use offilters, columns, flow tubes or channels with magnetic fields, etc., canthen be used to separate the target cell population from undesiredcells, debris, etc., that may be in a cellular sample. For example, atarget cell population can flow through a tube or other structure and beexposed to a magnetic field, whereby the target cell population isretained or held-up by the magnetic field, allowing undesired cells anddebris to pass through the tube. The magnetic field can then be turnedoff, allowing the target cell population to pass onto a furtherretention chamber or other area(s) of the cassette for further automatedprocessing. Additional filtration includes traditional columnfiltration, or use of other filtration membranes and structures.

In further embodiments, cassette 102 further includes a fixed volumewaste collection chamber 510 fluidly connected to tangential flow filter204. Fixed volume waste collection chamber 510 is used to collectpermeate flow 252 exiting the tangential flow filter. By utilizing afixed volume, the fixed volume waste collection chamber is allowed toonly hold a pre-determined about of collected permeate flow 252. Oncethis pre-determined amount of permeate flow 252 is reached, noadditional permeate flow 252 is allowed to exit tangential flow filter204, and thus the volume of the cellular sample will not be furtherreduced. This results in a cell concentration and cellular sample volumehaving a pre-defined and known value, for example, pre-defined to meetan end goal or for further processing of a defined volume. Examples offixed volume waste collection chambers 510 include various hardplastics, metals, etc., that will not expand and thus only hold a fixedvolume. In addition, a bag or flexible plastic can be used, but can beplaced inside of a hard plastic vessel or between non-moving walls(e.g., plastic walls), such that once the bag reaches a pre-determinedvolume, it impinges upon the non-moving walls or vessel, and theexpansion of the bag stops. As the fixed volume waste collection chamber510 fills to capacity, no additional permeate flow 252 is allowed toexit, and the retentate flow 254 then simply recirculates through thetangential flow filter, until such time as collection is desired.Suitably, this recirculation occurs via a fluidics pathway (i.e., showngenerically as 540 in the flowpath of FIG. 5. Fixed volume wastecollection chamber 510 can also include a level monitor that willtrigger and direct the permeate flow 252 to stop and recirculate theretentate flow 254.

In additional embodiments, a satellite volume 550, which can be provideadditional storage capabilities for the cassette, to increase theoverall volume of the automated processes, or additional volume flow forthe tangential flow filtration, is fluidly connected to tangential flowfilter 204. An exemplary location of satellite volume 550 is shown inthe flowpath of FIG. 5.

The cassettes can also further include one or more fluidics pathways(generically 540), wherein the fluidics pathways provide recirculation,removal of waste and homogenous gas exchange and distribution ofnutrients to various parts of the cassette, including the cell culturechamber without disturbing cells within the cell culture chamber.Cassette 102 also further includes one or more valves 522 or 552, forcontrolling the flow through the various fluidic pathways (see FIG. 5for exemplary locations within flowpath).

In exemplary embodiments, as shown in FIG. 2A, cell culture chamber 206is a flat and non-flexible chamber (i.e., made of a substantiallynon-flexible material such as a plastic) that does not readily bend orflex. The use of a non-flexible chamber allows the cells to bemaintained in a substantially undisturbed state. As shown in FIG. 2A,cell culture chamber 206 is oriented so as to allow the immune cellculture to spread across the bottom of the cell culture chamber. Asshown in FIG. 2A, cell culture chamber 206 is suitably maintained in aposition that is parallel with the floor or table, maintaining the cellculture in an undisturbed state, allowing the cell culture to spreadacross a large area of the bottom of the cell culture chamber. Inembodiments, the overall thickness of cell culture chamber 206 (i.e.,the chamber height) is low, on the order of about 0.5 cm to about 5 cm.Suitably, the cell culture chamber has a volume of between about 0.50 mland about 300 ml, more suitably between about 50 ml and about 200 ml, orthe cell culture chamber has a volume of about 180 ml. The use of a lowchamber height (less than 5 cm, suitably less than 4 cm, less than 3 cm,or less then 2 cm) allows for effective media and gas exchange in closeproximity to the cells. Ports are configured to allow mixing viarecirculation of the fluid without disturbing the cells. Larger heightstatic vessels can produce concentration gradients, causing the areanear the cells to be limited in oxygen and fresh nutrients. Throughcontrolled flow dynamics, media exchanges can be performed without celldisturbance. Media can be removed from the additional chambers (no cellspresent) without risk of cell loss.

As described herein, in exemplary embodiments the cassette is pre-filledwith one or more of a cell culture, a culture media, a cell wash mediaif desired, an activation reagent, and/or a vector, including anycombination of these. In further embodiments, these various elements canbe added later via suitable injection ports, etc.

As described herein, in embodiments, the cassettes suitably furtherinclude one or more of a pH sensor 524, a glucose sensor (not shown), anoxygen sensor 526, a carbon dioxide sensor (not shown), a lactic acidsensor/monitor (not shown), and/or an optical density sensor (notshown). See FIG. 5 for exemplary positions within the flowpath. Thecassettes can also include one or more sampling ports and/or injectionports. Examples of such sampling ports 220 and injection ports 222 areillustrated in FIG. 2A, and exemplary locations in the flowpath shown inFIG. 5, and can include an access port for connecting the cartridge toan external device, such as an electroporation unit or an additionalmedia source. FIG. 2A also shows the location of the input 202, reagentwarming bag 224 which can be used to warm cell media, etc., andsecondary chamber 230.

In embodiments, cassette 102 suitably includes a low temperaturechamber, which can include a refrigeration area 226 suitably for storageof a cell culture media, as well as a high temperature chamber, suitablyfor carrying out activation, transduction and/or expansion of a cellculture. Suitably, the high temperature chamber is separated from thelow temperature chamber by a thermal barrier. As used herein “lowtemperature chamber” refers to a chamber, suitably maintained below roomtemperature, and more suitably from about 4° C. to about 8° C., formaintenance of cell media, etc., at a refrigerated temperature. The lowtemperature chamber can include a bag or other holder for media,including about 1 L, about 2 L, about 3 L, about 4 L, or about 5 L offluid. Additional media bags or other fluid sources can be connectedexternally to the cassette, and connected to the cassette via an accessport.

As used herein “high temperature chamber” refers to chamber, suitablymaintained above room temperature, and more suitably maintained at atemperature to allow for cell proliferation and growth, i.e., betweenabout 35-40° C., and more suitably about 37° C. In embodiments, hightemperature chamber suitably includes cell culture chamber 206 (alsocalled proliferation chamber or cell proliferation chamber throughout).

In embodiments, tangential flow filter 204 is suitably aligned incassette 102 so that the tangential flow filter is at an angle of about3° to about 20°, relative to horizontal, more suitably about 5° to about15° or about 10°, relative to horizontal (exit end of tangential flowfilter 204 located above/higher than input end). Alignment of tangentialflow filter 204 at an angle relative to horizontal in which the exit end(i.e. 262) of tangential flow filter is above the input end is desirablefor providing the desired flow characteristics to yield improved volumereduction and cell concentration via tangential flow filter 204.

The alignment of the tangential flow filter at an angle between about 3°to about 20°, relative to horizontal, also provides the advantage thatcell priming (or gravity settling) can be reduced or avoided. Using suchan angle allows the cells tumble out of suspension as they flow down thetangential flow filter.

In embodiments, cassette 102 can also include a cell wash system 512that is suitably contained within cassette 102 (i.e., within thestructure shown in FIG. 2A), and fluidly connected to tangential flowfilter 204, or can be connected to other sections within the cassette,depending upon whether cell washing is desired. In embodiments, cellwash system 512 is a container or bag contained within cassette 102 thatsuitably includes a cell wash media. The cell wash media is suitablyused to clean the desired cell population to remove any undesired wastecells or contamination prior to transferring the cell population withinthe cassette or outside the cassette for further processing or use. Cellwash system 512 can also be included outside of cassette 102.

Cassette 102 can also further optionally include a cell holding chamber516 (not visible in FIG. 2 as it is located inside cassette 102). FIG. 5shows an exemplary location of cell holding chamber 516 in the flowpathfor the cassette. Cell holding chamber 516 is suitably a reservoir orsuitable chamber located within the cassette into which a cellpopulation can be held, either prior to or following tangential flowfiltration, as described herein.

In additional embodiments, provided herein is cassette 102 for use in anautomated cell engineering system 300, suitably comprising cell culturechamber 206, pumping system 520 fluidly connected to the cell culturechamber, and tangential flow filter 204 fluidly connected to the pumpingsystem. As described herein, the pumping system provides a retentateflow to the tangential flow filter and a permeate flow of the tangentialflow filter is controlled by a flow controller. The cassette alsofurther includes satellite volume 550 connected to the tangential flowfilter, a fluidics pathway 540 for recirculating the retentate flow backthrough the tangential flow filter, fixed volume waste collectionchamber 510 fluidly connected to the tangential flow filter, andcellular sample output 208 fluidly connected to the tangential flowfilter.

Exemplary pore sizes and fiber diameters for use in tangential flowfilter 204 are described herein. In embodiments, the tangential flowfilter has a pore size of about 0.40 μm to about 0.80 μm and a fiberdiameter of about 0.5 mm to about 0.9 mm, including a pore size of about0.60 μm to about 0.70 μm and a fiber diameter of about 0.70 mm to about0.80 mm.

Suitable materials for use in tangential flow filter include a polymer,such as but not limited to, poly(ether sulfone), poly(acrylonitrile) andpoly(vinylidene difluoride).

In exemplary embodiments, cassette 102 further includes one or morefluidics pathways, wherein the fluidics pathways provide recirculation,removal of waste and homogenous gas exchange and distribution ofnutrients to the cell culture chamber without disturbing cells withinthe cell culture chamber. In embodiments, the cell culture chamber is aflat and non-flexible chamber, having a low chamber height.

As described herein, cassette 102 can further include one or more of apH sensor, a glucose sensor, an oxygen sensor, a carbon dioxide sensor,and/or an optical density sensor, and can also include one or moresampling ports.

In embodiments, the tangential flow filter is at an angle of about 3° toabout 20°, relative to horizontal, located with cassette 102.

As described herein, the flow controller can be a flow restrictor, anadditional pumping system, a system having a plurality of tubing, orcombinations of such controllers.

FIGS. 3A-3B show the COCOON® automated cell engineering system 300 withcassette 102 positioned inside (cover of automated cell engineeringsystem opened in FIG. 3B). Also shown is an exemplary user interface,which can include a bar code reader, and the ability to receive usinginputs by touch pad or other similar device.

The automated cell engineering systems and cassettes described hereinsuitably have three relevant volumes, the cell culture chamber volume,the working volume, and the total volume. Suitably, the working volumeused in the cassette ranges from 180 mL to 460 mL based on the processstep, and can be increased up to about 500 mL, about 600 mL, about 700mL, about 800 mL, about 900 mL or about 1 L. In embodiments, thecassette can readily achieve 4*10⁹ cells-10*10⁹ cells. The cellconcentration during the process varies from 0.3*10⁶ cells/ml toapproximately 10*10⁶ cells/ml. The cells are located in the cell culturechamber, but media is continuously recirculated through additionalchambers (e.g., crossflow reservoir and satellite volume) to increasethe working volume, as described herein.

Fluidics pathways, including gas exchange lines, may be made from agas-permeable material such as, e.g., silicone. In some embodiments, theautomated cell engineering system recirculates oxygen throughout thesubstantially non-yielding chamber during the cell production methods.Thus, in some embodiments, the oxygen level of a cell culture in theautomated cell engineering system is higher than the oxygen level of acell culture in a flexible, gas-permeable bag. Higher oxygen levels maybe important in the cell culture expansion step, as increased oxygenlevels may support increased cell growth and proliferation.

In further embodiments, provided herein is a method of reducing a volumeof a cellular sample during automated processing. The method providedherein is described with reference to the flowpath of FIG. 5 forillustrative purposes only, but should not be considered to limit theway in which such a method can be carried out. For example, a cellularsample can be introduced into cassette 102 via input 202. In otherembodiments, a cellular sample can already be within cassette 102, forexample following a transduction or cell expansion phase, for example incell culture chamber 206. The cellular sample is introduced 250 intotangential flow filter 204, for example by passing through valve V11.The tangential flow filter has a retentate flow 254 and a permeate flow252 (see FIG. 2C). As described herein, permeate flow 252 is controlledby flow controller 258 to provide the desired cell concentration andvolume reduction. The cellular sample is passed through retentate flow254, while volume is removed from the cellular sample via permeate flow252. Suitably, permeate flow 252 is removed to fixed volume wastecollection chamber 510 by passing through valves v1 and v13 (thoughvalve v13 can be removed if desired). Once the desired reduction involume is attained, the cellular sample having the reduced volume iscollected, suitably by passing through valves V1 and V10 to output 208.In other embodiments, the cellular sample with the reduced volume can becollected in, for example, cell holding chamber 516, prior to furtherautomated processing or removal from the cassette.

As described herein, retentate flow 254 is suitably recirculatedfollowing the removing volume step to repeatedly pass the cellularsample through retentate flow 254. For example retentate flow 254 canpass out of tangential flow filter 204, through valves V1, V12 and V11,and back into tangential flow filter 204.

In embodiments that utilize fixed volume waste collection chamber 510,once a fixed volume of waste is reached, this will also force thecellular sample back through the tangential flow filter (e.g. throughvalves V14, V12 and V11), but will not allow any additional volumeremoval, as removing volume suitably stops once the fixed volume wastecollection chamber contains a desired volume.

In additional embodiments, following an initial collection of thecellular sample, the sample can be washed using cell wash system 512,and then the volume reduction method can be repeated. Cell wash system512 can be connected to cell holding chamber 516, for example, viavalves V4, and by closing valves V12 and V11, to force the wash solutioninto the holding chamber.

The methods described herein can further include additional steps,including for example electroporating the cellular sample followingcollecting after tangential flow filtration. This can occur via aninternal (i.e., with cassette 102) or external electroporation system.Additional transduction steps can also be carried out following thecollecting after tangential flow filtration.

As described herein, the methods suitably utilize a flow controller thatcan be a flow restrictor, an additional pumping system, a system havinga plurality of tubing, or combinations of such controllers.

In embodiments, the methods and cartridges described herein are utilizedin the COCOON® platform (Octane Biotech (Kingston, ON)), whichintegrates multiple unit operations in a single turnkey platform.Multiple cell protocols are provided with very specific cell processingobjectives. To provide efficient and effective automation translation,the methods described utilize the concept ofapplication-specific/sponsor-specific disposable cassettes that combinemultiple unit operations—all focused on the core requirements of thefinal cell therapy product. Multiple automated cell engineering systems300 can be integrated together into a large, multi-unit operation forproduction of large volumes of cells or multiple different cellularsamples for individual patients (see FIG. 4).

Also illustrated in FIG. 5 are exemplary positioning of various sensors(e.g., pH sensor 524, dissolved oxygen sensor 526), as well assampling/sample ports and various valves (including bypass check valves552), as well as one or more fluidic pathways 540, suitably comprising asilicone-based tubing component, connecting the components. As describedherein, use of a silicone-based tubing component allows oxygenationthrough the tubing component to facilitate gas transfer and optimaloxygenation for the cell culture. Also show in FIG. 5 is the use of oneor more hydrophobic filters 554 or hydrophilic filters 556, in theflowpath of the cassette.

In additional embodiments, provided herein is an automated cellengineering system 300. As shown in FIGS. 3A and 3B, automated cellengineering system 300 suitably includes an enclosable housing 302, andcassette 102, contained within the enclosable housing. As used herein,“enclosable housing” refers to a structure than can be opened andclosed, and within which cassette 102 as described herein, can be placedand integrated with various components such as fluid supply lines, gassupply lines, power, cooling connections, heating connections, etc. Asshown in FIGS. 3A and 3B, enclosable housing can be opened (FIG. 3B) toallow insertion of the cassette, and closed (FIG. 3A) to maintain aclosed, sealed environment to allow the various automated processesdescribed herein to take place utilizing the cassette.

As described herein, cassette 102 suitably includes cell culture chamber206, pumping system 520 fluidly connected to the cell culture chamber,and tangential flow filter 204 fluidly connected to the pumping system.As described herein, the pumping system provides a retentate flow to thetangential flow filter, and a permeate flow of the tangential flowfilter is controlled by a flow controller. Cassette 102 also suitablyincludes cellular sample output 208 fluidly connected to the tangentialflow filter.

As shown in FIGS. 3A-3B, automated cell engineering system 300 alsofurther includes a user interface 304 for receiving input from a user.User interface 304 can be a touch pad, tablet, keyboard, computerterminal, or other suitable interface, that allows a user to inputdesired controls and criteria to the automated cell engineering systemto control the automated processes and flowpath. Suitably, the userinterface is coupled to a computer control system to provideinstructions to the automated cell engineering system, and to controlthe overall activities of the automated cell engineering system. Suchinstructions can include when to open and close various valves, when toprovide media or cell populations, when to increase or decrease atemperature, etc.

Exemplary characteristics of the pore size and fiber diameter oftangential flow filter 204 for use in the automated cell engineeringsystems are described herein, and in embodiments, the tangential flowfilter has a pore size of about 0.40 μm to about 0.80 μm and a fiberdiameter of about 0.5 mm to about 0.9 mm, suitably a pore size of about0.60 μm to about 0.70 μm and a fiber diameter of about 0.70 mm to about0.80 mm. Suitably polymers for use in the tangential flow filter aredescribed herein, and include poly(ether sulfone), poly(acrylonitrile)and poly(vinylidene difluoride).

In embodiments, the cassette in the automated cell engineering systemsfurther comprises a fixed volume waste collection chamber 510 fluidlyconnected to the tangential flow filter 204. In embodiments, thecassettes 102 of the automated cell engineering systems 300 furtherinclude one or more fluidics pathways 540, wherein the fluidics pathwaysprovide recirculation, removal of waste and homogenous gas exchange anddistribution of nutrients to the cell culture chamber 206 withoutdisturbing cells within the cell culture chamber. In embodiments, thecell culture chamber is flat and non-flexible chamber, having a lowchamber height. Fluidics pathways can also be included for recirculatingthe retentate flow back through the tangential flow filter. Thecassettes can also include a satellite volume 550 fluidly connected tothe tangential flow filter.

In embodiments of the automated cell engineering system, the cassette102 is pre-filled with culture media, cell wash media, etc. As describedherein, in embodiments, the cassette of the automated cell engineeringsystem can further include one or more of a pH sensor 524, a glucosesensor, an oxygen sensor 526, a carbon dioxide sensor, and/or an opticaldensity sensor, and in suitable embodiments, one or more sampling ports.

Exemplary flow controllers are described herein, and include a flowrestrictor, an additional pumping system, and a system having aplurality of tubing. In embodiments, the tangential flow filter is at anangle of about 3° to about 20°, relative to horizontal, within thecassette.

Automation of unit operations in cell therapy production provides theopportunity for universal benefits across allogeneic and autologous celltherapy applications. In the unique scenario of patient-specific,autologous cell products, and even more emphasized by the clinicalsuccess of these therapies, the advantages of automation areparticularly compelling due to the significant micro-lot complexities ofsmall batch GMP compliance, economics, patient traceability and earlyidentification of process deviations. The associated emergence ofcomplex manufacturing protocols draws attention to the fact that thevalue of end-to-end integration of automated unit operations inmicro-lot cell production has not been a point of significant study.However, the expected demand for these therapies following theirimpending approval indicates that implementation of a fully closedend-to-end system can provide a much needed solution to manufacturingbottlenecks, such as hands-on-time and footprint.

Developers of advanced therapies are encouraged to consider automationearly in the rollout of clinical translation and scale up of clinicaltrial protocols. Early automation can influence protocol development,avoid the need for comparability studies if switching from a manualprocess to an automated process at a later stage, and provide a greaterunderstanding of the longer-term commercialization route.

In exemplary embodiments, the automated cell engineering systemsdescribed herein comprise a plurality of chambers, and wherein each ofsteps of the various methods described herein are performed in adifferent chamber of the plurality of chambers of the automated cellengineering system, each of the activation reagent, the vector, and cellculture medium are contained in a different chamber of the plurality ofthe chambers prior to starting the method, and wherein at least one ofthe plurality of chambers is maintained at a temperature for growingcells (e.g., at about 37° C.) and at least one of the plurality ofchambers is maintained at a refrigerated temperature (e.g., at about4-8° C.).

In embodiments, the automated cell engineering systems described hereinare monitored with a temperature sensor, a pH sensor, a glucose sensor,an oxygen sensor, a carbon dioxide sensor, and/or an optical densitysensor. Accordingly, in some embodiments, the automated cell engineeringsystem includes one or more of a temperature sensor, a pH sensor, aglucose sensor, an oxygen sensor, a carbon dioxide sensor, and/or anoptical density sensor. In additional embodiments, the automated cellengineering system is configured to adjust the temperature, pH, glucose,oxygen level, carbon dioxide level, and/or optical density of the cellculture, based on the pre-defined culture size. For example, if theautomated cell engineering system detects that the current oxygen levelof the cell culture is too low to achieve the necessary growth for adesired cell culture size, the automated cell engineering system willautomatically increase the oxygen level of the cell culture by, e.g.,introducing oxygenated cell culture media, by replacing the cell culturemedia with oxygenated cell culture media, or by flowing the cell culturemedia through an oxygenation component (i.e., a silicone tubing). Inanother example, if the automated cell engineering system detects thatthe current temperature of the cell culture is too high and that thecells are growing too rapidly (e.g., possible overcrowding of the cellsmay lead to undesirable characteristics), the automated cell engineeringsystem will automatically decrease the temperature of the cell cultureto maintain a steady growth rate (or exponential growth rate, asdesired) of the cells. In still further embodiments, the automated cellengineering system automatically adjusts the schedule of cell feeding(i.e., providing fresh media and/or nutrients to the cell culture) basedon the cell growth rate and/or cell count, or other monitored factors,such as pH, oxygen, glucose, etc. The automated cell engineering systemmay be configured to store media (and other reagents, such as washsolutions, etc.) in a low-temperature chamber (e.g., 4° C. or −20° C.),and to warm the media in a room temperature chamber or ahigh-temperature chamber (e.g., 25° C. or 37° C., respectively) beforeintroducing the warmed media to the cell culture.

Additional Exemplary Embodiments

Embodiment 1 is a cassette for use in an automated cell engineeringsystem, comprising a cell culture chamber, a pumping system fluidlyconnected to the cell culture chamber, a tangential flow filter fluidlyconnected to the pumping system, wherein the pumping system provides aretentate flow to the tangential flow filter and wherein a permeate flowof the tangential flow filter is controlled by a flow controller, and acellular sample output fluidly connected to the tangential flow filter.

Embodiment 2 includes the cassette of embodiment 1, wherein thetangential flow filter has a pore size of about 0.40 μm to about 0.80 μmand a fiber diameter of about 0.5 mm to about 0.9 mm.

Embodiment 3 includes the cassette of embodiment 2, wherein thetangential flow filter has a pore size of about 0.60 μm to about 0.70 μmand a fiber diameter of about 0.70 mm to about 0.80 mm.

Embodiment 4 includes the cassette of any one of embodiments 1-3,wherein the tangential flow filter comprises a polymer selected from thegroup consisting of poly(ether sulfone), poly(acrylonitrile) andpoly(vinylidene difluoride).

Embodiment 5 includes the cassette of any one of embodiments 1-4,further comprising a fixed volume waste collection chamber fluidlyconnected to the tangential flow filter.

Embodiment 6 includes the cassette of any one of embodiments 1-5,further comprising a fluidics pathway for recirculating the retentateflow back through the tangential flow filter.

Embodiment 7 includes the cassette of any one of embodiments 1-6,further comprising a satellite volume fluidly connected to thetangential flow filter.

Embodiment 8 includes the cassette of any one of embodiments 1-7,further comprising one or more fluidics pathways, wherein the fluidicspathways provide recirculation, removal of waste and homogenous gasexchange and distribution of nutrients to the cell culture chamberwithout disturbing cells within the cell culture chamber.

Embodiment 9 includes the cassette of any one of embodiments 1-8,wherein the cell culture chamber is a flat and non-flexible chamber,having a low chamber height.

Embodiment 10 includes the cassette of any one of embodiments 1-9,further comprising one or more of a pH sensor, a glucose sensor, anoxygen sensor, a carbon dioxide sensor, and/or an optical densitysensor.

Embodiment 11 includes the cassette of any one of embodiments 1-10,further comprising one or more sampling ports.

Embodiment 12 includes the cassette of any one of embodiments, whereinthe tangential flow filter is at an angle of about 3° to about 20°,relative to horizontal.

Embodiment 13 includes the cassette of any one of embodiments 1-12,wherein the flow controller is a flow restrictor.

Embodiment 14 includes the cassette of any one of embodiments 1-13,wherein the flow controller is an additional pumping system.

Embodiment 15 includes the cassette of any one of embodiments 1-14,wherein the flow controller is a system having a plurality of tubing.

Embodiment 16 is a cassette for use in an automated cell engineeringsystem, comprising a cell culture chamber, a pumping system fluidlyconnected to the cell culture chamber, a tangential flow filter fluidlyconnected to the pumping system, wherein the pumping system provides aretentate flow to the tangential flow filter and wherein a permeate flowof the tangential flow filter is controlled by a flow controller, asatellite volume connected to the tangential flow filter, a fluidicspathway for recirculating the retentate flow back through the tangentialflow filter, a fixed volume waste collection chamber fluidly connectedto the tangential flow filter, and a cellular sample output fluidlyconnected to the tangential flow filter.

Embodiment 17 includes the cassette of embodiment 16, wherein thetangential flow filter has a pore size of about 0.40 μm to about 0.80 μmand a fiber diameter of about 0.5 mm to about 0.9 mm.

Embodiment 18 includes the cassette of embodiment 17, wherein thetangential flow filter has a pore size of about 0.60 μm to about 0.70 μmand a fiber diameter of about 0.70 mm to about 0.80 mm.

Embodiment 19 includes the cassette of any one of embodiments 16-18,wherein the tangential flow filter comprises a polymer selected from thegroup consisting of poly(ether sulfone), poly(acrylonitrile) andpoly(vinylidene difluoride).

Embodiment 20 includes the cassette of any one of embodiments 16-19,further comprising one or more fluidics pathways, wherein the fluidicspathways provide recirculation, removal of waste and homogenous gasexchange and distribution of nutrients to the cell culture chamberwithout disturbing cells within the cell culture chamber.

Embodiment 21 includes the cassette of any one of embodiments 16-20,wherein the cell culture chamber is a flat and non-flexible chamber,having a low chamber height.

Embodiment 22 includes the cassette of any one of embodiments 16-21,further comprising one or more of a pH sensor, a glucose sensor, anoxygen sensor, a carbon dioxide sensor, and/or an optical densitysensor.

Embodiment 23 includes the cassette of any one of embodiments 16-22,further comprising one or more sampling ports.

Embodiment 24 includes the cassette of any one of embodiments 16-23,wherein the tangential flow filter is at an angle of about 3° to about20°, relative to horizontal.

Embodiment 25 includes the cassette of any one of embodiments 16-24,wherein the flow controller is a flow restrictor.

Embodiment 26 includes the cassette of any one of embodiments 16-25,wherein the flow controller is an additional pumping system.

Embodiment 27 includes the cassette of any one of embodiments 16-26,wherein the flow controller is a system having a plurality of tubing.

Embodiment 28 is a method of reducing a volume of a cellular sampleduring automated processing, the method comprising introducing acellular sample into a tangential flow filter having a retentate flowand a permeate flow, wherein the permeate flow is controlled by a flowcontroller, passing the cellular sample through the retentate flow ofthe tangential flow filter, removing volume from the cellular sample viathe permeate flow to a fixed volume waste collection chamber, andcollecting the cellular sample having the reduced volume.

Embodiment 29 includes the method of embodiment 28, further comprisingrecirculating the retentate flow following the removing volume step torepeatedly pass the cellular sample through the retentate flow.

Embodiment 30 includes the method of any one of embodiments 28-29,wherein the removing volume stops once the fixed volume waste collectionchamber contains a desired volume.

Embodiment 31 includes the method of any one of embodiments 28-30,further comprising washing the cellular sample following the collecting,and repeating steps (a)-(d) of the method.

Embodiment 32 includes the method of any one of embodiments 28-31,further comprising electroporating the cellular sample following thecollecting.

Embodiment 33 includes the method of any one of embodiments 28-32,wherein the flow controller is a flow restrictor.

Embodiment 34 includes the method of any one of embodiments 28-33,wherein the flow controller is an additional pumping system.

Embodiment 35 includes the method of any one of embodiments 28-34,wherein the flow controller is a system having a plurality of tubing.

Embodiment 36 is an automated cell engineering system, comprising anenclosable housing, a cassette contained within the enclosable housing,the cassette comprising a cell culture chamber, a pumping system fluidlyconnected to the cell culture chamber, a tangential flow filter fluidlyconnected to the pumping system, wherein the pumping system provides aretentate flow to the tangential flow filter and wherein a permeate flowof the tangential flow filter is controlled by a flow controller, and acellular sample output fluidly connected to the tangential flow filter,and a user interface for receiving input from a user.

Embodiment 37 includes the automated cell engineering system ofembodiment 36, wherein the tangential flow filter has a pore size ofabout 0.40 μm to about 0.80 μm and a fiber diameter of about 0.5 mm toabout 0.9 mm.

Embodiment 38 includes the automated cell engineering system ofembodiment 37, wherein the tangential flow filter has a pore size ofabout 0.60 μm to about 0.70 μm and a fiber diameter of about 0.70 mm toabout 0.80 mm.

Embodiment 39 includes the automated cell engineering system of any oneof embodiments 36-38, wherein the tangential flow filter comprises apolymer selected from the group consisting of poly(ether sulfone),poly(acrylonitrile) and poly(vinylidene difluoride).

Embodiment 40 includes the automated cell engineering system of any oneof embodiments 36-39, further comprising a fixed volume waste collectionchamber fluidly connected to the tangential flow filter.

Embodiment 41 includes the automated cell engineering system of any oneof embodiments 36-40, further comprising a fluidics pathway forrecirculating the retentate flow back through the tangential flowfilter.

Embodiment 42 includes the automated cell engineering system of any oneof embodiments 36-41, further comprising a satellite volume fluidlyconnected to the tangential flow filter.

Embodiment 43 includes the automated cell engineering system of any oneof embodiments 36-42, further comprising one or more fluidics pathways,wherein the fluidics pathways provide recirculation, removal of wasteand homogenous gas exchange and distribution of nutrients to the cellculture chamber without disturbing cells within the cell culturechamber.

Embodiment 44 includes the automated cell engineering system of any oneof embodiments 36-43, wherein the cell culture chamber is a flat andnon-flexible chamber, having a low chamber height.

Embodiment 45 includes the automated cell engineering system of any oneof embodiments 36-44, further comprising one or more of a pH sensor, aglucose sensor, an oxygen sensor, a carbon dioxide sensor, and/or anoptical density sensor.

Embodiment 46 includes the automated cell engineering system of any oneof embodiments 36-45, further comprising one or more sampling ports.

Embodiment 47 includes the automated cell engineering system of any oneof embodiments 36-46, wherein the tangential flow filter is at an angleof about 3° to about 20°, relative to horizontal.

Embodiment 48 includes the automated cell engineering system of any oneof embodiments 36-47, further comprising a computer control system,wherein the user interface is coupled to the computer control system toprovide instructions to the automated cell engineering system.

Embodiment 49 includes the automated cell engineering system of any oneof embodiments 36-48, wherein the flow controller is a flow restrictor.

Embodiment 50 includes the automated cell engineering system of any oneof embodiments 36-48, wherein the flow controller is an additionalpumping system.

Embodiment 51 includes the automated cell engineering system of any oneof embodiments 36-48, wherein the flow controller is a system having aplurality of tubing.

EXAMPLES Example 1—Tangential Flow Filtration in COCOON™ System

Tangential flow filtration (TFF) for cell therapy applications can beused to separate, clarify, recover and collect cells from a post-harvestsuspension fluid, prior to formulation. A traditional TFF processconsists of two steps; 1) volume reduction and 2) diafiltration. Duringthe volume reduction step the bulk volume (cells in harvest reagent andculture media) is constantly removed via filtration through the permeateside of the filter until a desired cell concentration is reached in theprocessing bag. During diafiltration, the concentrated cell suspensionsolution is replaced with a formulation buffer and residual proteins andcontaminants that are undesirable in the final solution are reduced toacceptable levels. The final cell suspension will be at a cellconcentration and in a buffer that is ready for formulation. Tangentialflow filters are preferred over standard filters as they can reducefluid volume while preventing clogging and avoiding cell damage. Thecells are also easier to retrieve as they are not compressed against thefilter.

TFF filters are single use and disposable so they can be easilyimplemented into a cassette to perform operations in a closed andautomated manner. A completely closed system allows the process to beperformed aseptically, as cell therapy products cannot be terminallysterilized or filtered. A fully disposable system eliminatescross-contamination risks and reduces cleaning requirements. To increasethe functionality of the COCOON™, a cassette is provided with anintegrated tangential flow filter. This example details the developmentof a TFF system to concentrate and wash cells in an automated system forcell therapy applications.

Methods Tangential Flow Filtration in COCOON™ Cassette

TFF systems for cell concentration typically have two pumps, one tocontrol the feed flow rate and one to control the permeate (i.e. waste)flow rate. The flow rate of each pump is typically determined based onoptimizing transmembrane pressure. If the pressure differential is toohigh or too low, it can cause either nothing to pass through the filter,thus making the system ineffective, or it can lead to clogging. TheCOCOON™ generally operates on a single pump, and without pressuresensors, so the conventional methods of filtering via TFF do not apply.

Experiments were run with TFF filters installed in either a COCOON™cassette or cassette-like pathway. As described herein, the cassettepathway contains an expansion chamber for cell culture, satellite bagsor L-shaped chamber for cell processing, a TFF to remove excess media,and a waste bag to collect the excess media. The COCOON™ cassettesuitably recirculates up to 450 mL of culture media in its culturechamber. Additional media volume beyond the 180 mL capacity of the 260cm² proliferation chamber is provided from various satellite reservoirsof the COCOON™ cassette. The additional media from these satellitereservoirs can be recirculated within the culture portion of thedisposable cassette to provide fresh nutrients and remove waste productsfrom cells in the proliferation chamber.

To generate a pressure differential, a flow restrictor was used in thepermeate line. Based on experimental optimization, an ideal permeateflow rate was selected that avoided clogging, maximized cell recoveryand minimized time for the volume reduction. In parallel with this, awide range of filters were tested to understand the impact of fiberdiameter, fiber area, number of fibers, total surface area, cell type,retentate flow rate, pore size and filter material.

Fixed Volume Waste Container

Several experiments also utilized a fixed volume waste container.Cassettes typically have a flexible waste bag located in the fluidsreservoir. This bag has the capacity to expand, potentially leading tocomplete drain of the satellite bag and TFF in certain situations. Acomplete drain of the filter leads to irreversible loss of cells due totrapping on the filter membrane. To limit the capacity of the waste bag,it can be held between two rigid layers of plastic with fixed separationin the fluids reservoir. The bag fills to a fixed volume, at which pointthe pressure in the bag is such that recirculation through the satellitebag/TFF continues, without further delivery of fluid to waste.

Custom Filter to Concentrate Peripheral Blood Mononuclear Cells (PBMC)

Initial cell concentration experiments revealed desired properties of atangential flow filter, such as increased surface area and a large poresize. The Spectrum Labs P-OCTA01-04-N filter is a custom designed filterto meet these requirements and fit inside a Cocoon cassette. Propertiesinclude:

mPES membrane

Fiber diameter=0.75

Pore size=0.65 μm

Number of Fibers=18

Lumen=13 cm total length

Surface area=57 cm²

This filter was evaluated, optimized, and then used in proof-of-conceptelectroporation integration studies.

TFF Volume Reduction Using Custom Filter

Initial studies of the custom filter were performed without the COCOON™.The KrosFlo® Research 2 i TFF System (Spectrum Labs) was used to monitorfeed, retentate, permeate and transmembrane pressures during cellprocessing. Only one pump that controls the flow rate of the feed linewas utilized (unless mentioned otherwise) to mimic the COCOON™instrument capabilities. A 20 gauge, 0.024″ I.D./0.036″ O.D., flowrestrictor from Nordson EFD, which was added to the end of the permeateline to mimic previously optimized TFF procedures. By using this system,a cell suspension of 100 mL was concentrated down to 10-20 mL. TFF wasperformed on the benchtop at room temperature. Transmembrane pressure isdefined as:

${TMP} = {\frac{P_{feed} + {Pretentat}}{2} - {Ppermeate}}$

PBMC Culture

1×10⁸ PBMCs were stimulated with 1×10⁸ CD3+:CD28+ Dynabeads (Invitrogen)and expanded in Complete T-cell Media comprised of X-VIVO 15 media(Lonza) supplemented with 5% Human Serum A/B (Sigma) and 10 ng/mL IL-2(Peprotech) using multiple GREX 100 (Wilson Wolf) culture vessels for upto 10 days. To accommodate high viscosity serum that can clog thefilter, a pre-wash protocol in the COCOON™ has been defined to firstreduce the concentration of the serum prior to the volume reductionusing the TFF process. Test concentrations of cells were transferred to250 mL conical vials and either centrifuged or allowed to settle in 37°C. incubators with 5% CO₂ in air humidified for 2-4 hrs. The supernatantof the settled cell suspension was reduced to 10 mL and excesssupernatant discarded. The appropriate media was added to theconcentrated cell suspension for a final volume of 100 mL.

Analysis

Counts were performed in duplicate using the Nucleocounter NC-200(Chemometec) on the pre-diluted cell culture, the diluted culture andthe final concentrated cell suspension. Volumes were measured using aserological pipette and KrosFlo scales before and after TFF. Residualtesting samples were obtained from the initial culture pre-dilution,supernatant pre-TFF, and final concentrated cell suspension post TFF. AHuman Serum ELISA Kit (Bethyl Laboratories) was used to determine thepercentage of serum remaining post dilution and concentration. FACSanalysis was performed on control cells and TFF concentrated cellsuspensions for CD4+ and CD8+ expression.

Successful demonstration of TFF volume reduction was defined as follows:

≥85% recovery of cells post TFF

≤10% decrease in cell viability post TFF

≤10%, Residual human serum of the initial concentration post TFF (forelectroporation studies)

Results Evaluation of Tangential Flow Filters

A wide variety of filters were tested to understand the impact ofvarious filter parameters. Fiber diameter, fiber area, number of fibers,total surface area, retentate flow rate, pore size, and filter materialall play a role in the effectiveness of the filter in reducing thevolume of a cell suspension. The results were also impacted by thesolution (e.g. media type, and the type of serum) as well as the celltype (i.e. size), the number of cells, the cell concentration and thetarget final volume. Hydrostatic pressure was also influential and sothe flow restrictor had to be adjusted depending on hydrostaticpressure. Most runs used human mesenchymal stem cells (hMSC) as thetested cell type.

To accommodate variability in the amount of permeate flow, anon-flexible waste container was used with a fixed volume. For example,if 100 mL needed to be removed from the total volume, a waste containerof exactly 100 mL was used. The duration of flow could be set based onthe slowest permeate flow. A by-pass loop was placed on either side ofthe pump tube with an in-line high pressure check valve. If the wastefilled before the pumping time was complete, the by-pass line wasactivated, causing the fluid to pump in a circle, thus ending the TFFprocess. This approach achieved very consistent flow rate to waste asdemonstrated in Table 1. For additional control, a level sensor can beintegrated into the COCOON™ to monitor the fluid level in thenon-flexible container.

TABLE 1 Fixed Volume Waste Container Run Summary Control CalculatedConcentrated Post- Total Post-Test Overall Starting Volume ChangeCollection Cell Calculated a % of Efficiency Number Suspension CellWaste (Including Final in Viability Recovery Overall starting adjusted(Live Volume Viability Volume 8 mL rinse) Viability Viability Control(Live Efficiency population for control # M) (mL) (%) (mL) (mL) (%) (%)(%) # M) (%) (%) (%) 40 160 97.7 123 35.7 92.2 −5.5 — 29.7 74.3 — — 40160 95 117 35.8 93.1 −1.9 94.8 29.2 72.9 89.8 83 38 160 94.9 117 38.593.8 −1.1 94.9 32.7 86.8 94.9 82 40 160 95.1 119 34.1 90.2 −4.9 96.629.3 73.0 88.7 83 39 160 94.4 120 34.9 88.8 −5.6 92.9 27.7 70 86.3 80 38160 93.7 120 35.7 89.8 −3.9 92.5 27.9 74 88.8 82 41 160 96 124 35.3 89.8−6.2 96.3 31.2 76.4 91.2 83 37 160 95.6 120 43 94.1 −1.2 94.7 29.5 8088.2 89 38 160 98.4 125 36.2 95.3 −3.1 97.2 28.7 76 92.7 82 38 160 97.8126 30.7 94.5 −3.3 95.1 30.7 81.3 97.8 83

Evaluation and Optimization of Custom Tangential Flow Filter

The results of the testing of the various tangential flow filtersrevealed desirable conditions. A custom filter, Spectrum LabsP-OCTA01-04-N, met these specifications, but testing and optimizationwas needed. We wanted to ensure that the filter was working correctlyand initially decouple any limitations of the COCOON™ system; therefore,we used a Spectrum Labs TFF system to evaluate the filter.

Acellular runs were initiated to receive initial working parameters ofthe filter. When the volume of RPMI media was reduced, there was aconstant transmembrane pressure (TMP) and flux through the filter (FIG.6A). However, if serum is added to the RMPI, TMP increases and fluxdecrease over time (FIG. 6B). This is a sign that the filter is cloggingfrom the proteins in the serum.

To control the clogging from the serum, either an automated backpressurevalve (FIGS. 7A and 7B) or secondary pump (FIG. 7C) was added to thepermeate line. The automated backpressure valve is able to control thepermeate pressure after 3 minutes of volume reduction. The secondarypump controlled the permeate flowrate to 20 ml/min initially and then 10ml/min after 5.5 minutes. In both cases of permeate control, there was amostly constant flux, permeate pressure, and TMP. The results indicatethat controlling the pressure on the TFF permeate line in the COCOON™,provides control over filter clogging.

Similar trends are seen with the volume reduction of PBMC suspensionswithout serum (FIGS. 8A and 8B). The addition of a backpressure controlvalve on the permeate helps stabilize flux, permeate pressure and TMP.This further confirms the need for permeate control.

In order to receive the greatest cell recovery without significant lossin viability, process parameters are optimized. The first parameterexamined is the permeate pressure via the permeate control pump. Whileconcentrating PMBC+0% serum suspensions, the recirculation pump was setto 60 mL/min and the permeate control pump was set to either 0, 5, 10,or 15 mL/min (FIGS. 9A-9D). Speed of the permeate pump appeared to havelittle effect on the flux, TMP or permeate pressure. 15 mL/min waschosen for the following experiments as this will lead to the quickestTFF duration.

Recirculation flow rate was also examined. PBMCs in a 0% serumsuspension were concentrated by TFF with the permeate pump at 15 mL/minand the recirculation flow rate at either 60 mL/min and 70 mL/min (Table2). There was a larger recovery of cells with a flowrate of 70 mL/min.

TABLE 2 Tangential Flow Filtration Concentration of PMBCs with 0% Serumfor Permeate Control Recirculation Initial Final Final Initial Flow rateVolume Volume Viability cell % Trial (mL/min) (mL) (mL) (%) countRecovery 1 60 103 16 93 1.8E9 78.6 2 70 100 15.5 94 1.5E9 95.5 3 70 10015.5 96 1.5E9 95.5

PBMCs in a 0% serum suspension were concentrated by TFF with a flowrestrictor on the permeate line and a recirculation flow rate of 70mL/min (Table 3). The average recovery was approximately 89% withviabilities greater than 80%.

TABLE 3 Tangential Flow Filtration Concentration of PMBCs with 0% Serumand Flow Restrictor Initial Final Initial Final Initial Volume VolumeViability Viability cell % Trial (mL) (mL) (%) (%) count Recovery 1 10120 97 96 2.2E9 85 2 100 17.8 96 96 1.9E9 93 3 103 18 96 96 2.8E9 93 4100 20 96 95 2.6E9 96.6 5 104 17.5 90 84 3.4E9 76 average 93.4 88.7

Many cellular therapies use serum, and in some instances, it may not bepossible to remove serum prior to TFF. PBMCs in a 5% serum suspensionwere concentrated by TFF with a flow restrictor on the permeate line andthe recirculation flow of 70 mL/min (Table 4). The average recovery wasapproximately 86% with viabilities greater than 80%.

TABLE 4 Tangential Flow Filtration Concentration of PMBCs with 5% Serumand Flow Restrictor Initial Final Initial Final Initial Volume VolumeViability Viability cell % Trial (mL) (mL) (%) (%) count Recovery 1 10017.5 96 98 1.8E9 95 2 100 20.3 97 79 4.5E9 75.7 3 100 20.5 97 90 2.96E9 89.5 4 100 19.5 97 85 2.4E9 84.4 average 87.5 86

Cell Concentration in the COCOON™ Cassette Via TFF for Electroporation

Cell wash and concentration is not only useful prior to downstreamprocessing of a product; it can also be utilized mid-automated processfor certain unit operations such as electroporation. Before cells areadded to an electroporation unit, cells are suitably concentrated to <10mL, and residuals washed out. For a proof-of-concept, cells from twodonors were concentrated by settling to a 10 mL volume with 4.4×10⁸ and4.2×10⁸ total viable cells. These two cell suspensions were then dilutedwith 90 mL of supplemented Nucleofector™ Solution (NFS) and concentratedto 10 mL using TFF. Cell recovery post TFF concentration was 92% and 87%(FIG. 10A). Cell viability prior to transfection was 92% and 74% anddecreased by less than 5% post TFF (FIG. 10B).

In both runs, 6% and 8% of the initial culture supernatant was detectedin the final TFF concentrated cell suspension (Table 5).

TABLE 5 Percentage of detectable human serum A/B in the original culturesupernatant, post diluted and concentrated TFF permeate, and final cellsuspension supernatant post TFF. Human Serum Human Human Human HumanConcen- Serum Serum Serum Serum tration of Concen- Concen- Concen-Concen- Initial tration tration tration tration Sample Culture Pre-TFFPre-TFF Post TFF Post TFF ID (ng/mL) (ng/mL) (% of initial) (ng/mL) (%of initial) Donor 1 4.98E+6 2.19E+5 4%  2.8E+5 6.00% Donor 2 4.28E+63.48E+5 9% 3.30E+5 8.20%

There was no difference in CD4+:CD8+ profiles post TFF compared to thecontrol culture that was not concentrated by TFF (FIG. 11).

These results demonstrate the use of TFF in the washing andconcentration of cells prior to in-process transfection.

CONCLUSION

Wash and concentration via Tangential Flow Filtration can be suitablycarried out using the COCOON™ system. TFF allows processes to remainclosed and automated and fits within the confines of a COCOON™disposable cassette. TFF can concentrate cell suspensions <20 ml andrecover >85% of cells through the system.

It will be readily apparent to one of ordinary skill in the relevantarts that other suitable modifications and adaptations to the methodsand applications described herein can be made without departing from thescope of any of the embodiments.

It is to be understood that while certain embodiments have beenillustrated and described herein, the claims are not to be limited tothe specific forms or arrangement of parts described and shown. In thespecification, there have been disclosed illustrative embodiments and,although specific terms are employed, they are used in a generic anddescriptive sense only and not for purposes of limitation. Modificationsand variations of the embodiments are possible in light of the aboveteachings. It is therefore to be understood that the embodiments may bepracticed otherwise than as specifically described.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by reference.

1. A cassette for use in an automated cell engineering system,comprising: (a) a cell culture chamber; (b) a pumping system fluidlyconnected to the cell culture chamber; (c) a tangential flow filterfluidly connected to the pumping system, wherein the pumping systemprovides a retentate flow to the tangential flow filter and wherein apermeate flow of the tangential flow filter is controlled by a flowcontroller; and (d) a cellular sample output fluidly connected to thetangential flow filter.
 2. The cassette of claim 1, wherein thetangential flow filter has a pore size of about 0.40 μm to about 0.80 μmand a fiber diameter of about 0.5 mm to about 0.9 mm.
 3. The cassette ofclaim 2, wherein the tangential flow filter has a pore size of about0.60 μm to about 0.70 μm and a fiber diameter of about 0.70 mm to about0.80 mm.
 4. The cassette of claim 1, wherein the tangential flow filtercomprises a polymer selected from the group consisting of poly(ethersulfone), poly(acrylonitrile) and poly(vinylidene difluoride).
 5. Thecassette of claim 1, further comprising a fixed volume waste collectionchamber fluidly connected to the tangential flow filter.
 6. The cassetteof claim 1, further comprising a fluidics pathway for recirculating theretentate flow back through the tangential flow filter.
 7. The cassetteof claim 1, further comprising a satellite volume fluidly connected tothe tangential flow filter.
 8. The cassette of claim 1, furthercomprising one or more fluidics pathways, wherein the fluidics pathwaysprovide recirculation, removal of waste and homogenous gas exchange anddistribution of nutrients to the cell culture chamber without disturbingcells within the cell culture chamber.
 9. The cassette of claim 1,wherein the cell culture chamber is a flat and non-flexible chamber,having a low chamber height.
 10. The cassette of claim 1, furthercomprising one or more of a pH sensor, a glucose sensor, an oxygensensor, a carbon dioxide sensor, and/or an optical density sensor. 11.The cassette of claim 1, further comprising one or more sampling ports.12. The cassette of claim 1, wherein the tangential flow filter is at anangle of about 3° to about 20°, relative to horizontal.
 13. The cassetteof claim 1, wherein the flow controller is a flow restrictor.
 14. Thecassette of claim 1, wherein the flow controller is an additionalpumping system.
 15. The cassette of claim 1, wherein the flow controlleris a system having a plurality of tubing.
 16. A cassette for use in anautomated cell engineering system, comprising: (a) a cell culturechamber; (b) a pumping system fluidly connected to the cell culturechamber; (c) a tangential flow filter fluidly connected to the pumpingsystem, wherein the pumping system provides a retentate flow to thetangential flow filter and wherein a permeate flow of the tangentialflow filter is controlled by a flow controller; (d) a satellite volumeconnected to the tangential flow filter; (e) a fluidics pathway forrecirculating the retentate flow back through the tangential flowfilter; (f) a fixed volume waste collection chamber fluidly connected tothe tangential flow filter; and (g) a cellular sample output fluidlyconnected to the tangential flow filter.
 17. The cassette of claim 16,wherein the tangential flow filter has a pore size of about 0.40 μm toabout 0.80 μm and a fiber diameter of about 0.5 mm to about 0.9 mm. 18.The cassette of claim 17, wherein the tangential flow filter has a poresize of about 0.60 μm to about 0.70 μm and a fiber diameter of about0.70 mm to about 0.80 mm.
 19. The cassette of claim 16, wherein thetangential flow filter comprises a polymer selected from the groupconsisting of poly(ether sulfone), poly(acrylonitrile) andpoly(vinylidene difluoride).
 20. The cassette of claim 16, furthercomprising one or more fluidics pathways, wherein the fluidics pathwaysprovide recirculation, removal of waste and homogenous gas exchange anddistribution of nutrients to the cell culture chamber without disturbingcells within the cell culture chamber.
 21. The cassette claim 16,wherein the cell culture chamber is a flat and non-flexible chamber,having a low chamber height.
 22. The cassette of claim 16, furthercomprising one or more of a pH sensor, a glucose sensor, an oxygensensor, a carbon dioxide sensor, and/or an optical density sensor. 23.The cassette of claim 16, further comprising one or more sampling ports.24. The cassette of claim 16, wherein the tangential flow filter is atan angle of about 3° to about 20°, relative to horizontal.
 25. Thecassette of claim 16, wherein the flow controller is a flow restrictor.26. The cassette of claim 16, wherein the flow controller is anadditional pumping system.
 27. The cassette of claim 16, wherein theflow controller is a system having a plurality of tubing.
 28. A methodof reducing a volume of a cellular sample during automated processing,the method comprising: (a) introducing a cellular sample into atangential flow filter having a retentate flow and a permeate flow,wherein the permeate flow is controlled by a flow controller; (b)passing the cellular sample through the retentate flow of the tangentialflow filter; (c) removing volume from the cellular sample via thepermeate flow to a fixed volume waste collection chamber; and (d)collecting the cellular sample having the reduced volume.
 29. Anautomated cell engineering system, comprising: (a) an enclosablehousing; (b) a cassette contained within the enclosable housing, thecassette comprising: i. a cell culture chamber; ii. a pumping systemfluidly connected to the cell culture chamber; iii. a tangential flowfilter fluidly connected to the pumping system, wherein the pumpingsystem provides a retentate flow to the tangential flow filter andwherein a permeate flow of the tangential flow filter is controlled by aflow controller; and iv. a cellular sample output fluidly connected tothe tangential flow filter; and (c) a user interface for receiving inputfrom a user.